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From the Departments of Pathology* and Medicine, and the W. Harry Feinstone Department of Molecular Microbiology and Immunology, the Johns Hopkins Medical Institutions, Baltimore, Maryland
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Abstract |
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Phenotypic characterization of the myocardial infiltrate has been traditionally assessed by immunohistochemical methods.7-9 Immunohistochemistry provides insights into the spatial distribution of the cellular infiltrate in the context of preserved cardiac morphology. However, the method is cumbersome for the purposes of quantifying cellular subsets and typically involves the assessment of one cellular marker at a time. Furthermore, the characterization of the infiltrate is limited to the chosen cross-section of the heart, which may not necessarily be representative of the whole myocardium because of the frequently patchy nature of myocarditis. These drawbacks necessitated the development of a more robust and quantitative method.
Flow cytometry represents a method of quantitative characterization of individual cells and provides a multiparametric assessment of cellular phenotypes by simultaneous cellular staining with multiple fluorochrome-labeled antibodies. It has been commonly applied for the analysis of peripheral blood, spleen, lymph nodes, and thymus, which was facilitated by the ease of individual cell isolation from these organs. The heart, however, presents a challenge in terms of cell separation. The myocardium represents a functional syncytium formed by anastomosing cardiomyocytes, which are supported and tethered by complex cardiac interstitium.10,11 These features of the myocardium make it difficult to achieve a uniform digestion with a satisfactory yield of infiltrating cells by simply placing myocardial tissue in a shaker with digestion solution, a method used for isolation of the inflammatory cells from other organs.12,13 These methodological difficulties have resulted in a limited use of flow cytometry in cardiac research. Meanwhile, in vitro studies of cardiac function have relied on cardiomyocyte isolation by means of retrograde perfusion of the coronary circulation with digestion solution.14,15 In this work, flow cytometry was used for the analysis of single cell suspensions obtained by cardiomyocyte isolation technique applied to individual murine hearts. Using this method, we assessed both the magnitude and phenotype of the cardiac infiltrate in normal and cardiac myosin (CM)-immunized mice. Finally, we combined flow cytometry with pressure-volume assessment16,17 to study the relationship between the nature of the inflammatory response and cardiac function in the chronic phase of experimental autoimmune myocarditis (EAM).
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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EAM was induced in 7- to 8-week-old male A/J mice and in 10- to 12-week-old female BALB/c mice obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in The Johns Hopkins University School of Medicine conventional animal facility. The animal work was approved by the Animal Care and Use Committee of The Johns Hopkins University.
Induction and Gross Assessment of EAM
CM was purified according to the procedure of Shiverick and colleagues18
from pooled hearts of different mouse strains, 60% of which were from susceptible BALB/c and A/J mice. On days 0 and 7, BALB/c mice received subcutaneous injections of CM (200 µg on day 0 and 250 µg on day 7) and A/J mice received 100 nmol of peptide derived from the 
-CM heavy chain19
[myhc(334-352) NH2-DSAFDVLSFTAEEKAGVYK-COOH, synthesized by Macromolecular Resources, Colorado State University, Department of Biochemistry, Fort Collins, CO] emulsified in complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO). When used in A/J mice, complete Freund’s adjuvant was supplemented with 5 mg/ml of killed Mycobacterium tuberculosis strain H37Ra (Difco, Detroit, MI). On day 0, mice received an intraperitoneal injection of 500 ng of pertussis toxin (List Biological Laboratories, Campbell, CA). Hearts were scored grossly based on the extent of white discoloration by two independent investigators using the following classification: grade 0, no disease; grade 1, up to 10% of heart surface discolored; grade 2, 11 to 30%; grade 3, 31 to 50%; grade 4, 50 to 90%; and grade 5, more than 90%.
Cell Isolation
Mice were injected with 1000 U of heparin 10 minutes before cervical dislocation. The heart was rapidly removed, placed in a 37°C water bath, and the aorta was cannulated with a 22-gauge needle connected to a modified Langendorff preparation. The heart was perfused at a constant flow of 1.1 ml/minute at 37°C for 3 minutes with a Ca2+-free bicarbonate-based buffer containing (in mmol/L) 120 NaCl, 5.4 KCl, 1.2 NaH2PO4, 20 NaHCO3, 5.6 glucose, 5 taurine, 1.6 MgCl2, and 10 2,3-butanedione monoxime. Enzymatic digestion was initiated by perfusion with the above solution with the addition of collagenase type 2 and protease type XIV (0.895 mg/ml and 0.5 mg/ml respectively; Boehringer Mannheim, Indianapolis, IN) for an additional 7 minutes. All solutions were equilibrated with a 95% O2-5% CO2 mixture for 20 minutes before use. The heart was placed into a Petri dish containing chilled staining buffer (1% fetal calf serum, 0.05% sodium azide in phosphate-buffered saline) and manually dispersed into a single cell suspension using razor blades. Single cell suspensions were sequentially filtered through 40-µm and 15-µm cell strainers. All reagents were obtained from Sigma-Aldrich except where indicated.
Flow Cytometry
Cells from individual hearts were resuspended in 50 ml of staining buffer, centrifuged at 352 x g for 8 minutes, and split into approximately five samples per heart based on cell counting so that each sample contained 
106 live (trypan blue-negative) cells in 100 µl of staining buffer. The mean and SD for live cell recoveries from individual mouse hearts were (7.2 ± 4.0) x 106/heart. Cells were preblocked with anti-Fc
receptor III/II monoclonal antibody (mAb) (clone 2.4G2) for 5 minutes and stained at +4°C for 30 minutes simultaneously with four colors using the following mAbs: fluorescein isothiocyanate-labeled anti-CD3 (clone 145-2C11), anti-CD44 (clone IM7), and Gr1 (clone RB6-8C5); phycoerythrin-labeled anti-CD4 (clone H129.19), anti-CD19 (clone 1D3), anti-MHC class II (clone NIMR-4), and anti-CD11b (clone M1/70); peridinin chlorophyll- protein-labeled anti-CD45 (clone 30-F11); allophycocyanin-labeled anti-CD3 (clone 145-2C11), and anti-CD8 (clone 53-6.7). Cells were washed, resuspended in 450 µl of staining buffer, and immediately analyzed. All reagents were from BD PharMingen (San Diego, CA), except for anti-MHC class II mAb, which was from Southern Biotechnology Associates, Inc. (Birmingham, AL). Data were acquired until 50,000 events were collected from the live gate using forward/side scatter plots. The live gate was set to exclude events with low forward scatter signals (Figure 1A)
. Cell fluorescence was measured with FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Data analysis was performed using FCS Express2 (De Novo Software, Richmond Hill, Ontario, Canada). Heart cell isolation and flow cytometry were performed on 6 to 10 mice per day.
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Pressure-volume studies were performed in immunized BALB/c mice on days 58 to 61 after immunization and in age-matched unimmunized controls following a previously established protocol.17
Anesthesia was initiated with methoxyflurane followed by an intraperitoneal injection of urethane (800 to 1000 mg/kg), etomidate (20 to 30 mg/kg), and morphine (1 to 2 mg/kg). After placing the animal on a heating pad, a tracheotomy was performed, the trachea was cannulated with a smoothed 19-gauge needle, and ventilation was initiated with 100% O2 at a tidal volume of 200 µl and a rate of 140 breaths/minute. A custom designed ventilator provided constant flow at peak airway pressures of 5 to 10 cmH2O. The left external jugular vein was exposed and cannulated using a 31-gauge needle. Volume expansion was achieved by infusion of 150 µl of 12.5% human albumin. The left ventricular apex was exposed via a subdiaphragmatic incision. An apical stab was made with a 26-gauge needle and a 1.4-F pressure-volume catheter (SPR-719; Millar, Houston, TX), containing four electrodes and a micromanometer, was placed to span the long axis of the left ventricle, such that the proximal electrode is just inside the endocardium and the distal electrode is just above or at the aortic valve. The catheter occupies a total volume of 0.76 µl and 20% of the cross-sectional area of the aortic valve at the widest part. Insertion of the pressure sensor beyond the valve confirms the absence of obstruction. The catheter was connected to a custom-designed signal conditioner, which provided a constant current of 30 µA at a frequency of 20 kHz. Parallel conductance (offset) was estimated using the hypertonic saline dilution method. Measurements of pressure and volume were obtained at steady state and during the preload reduction by transiently occluding the inferior vena cava. After all measurements were made, the mouse was placed on its side, a limited lateral thoracotomy was performed and the descending aorta was dissected free from the spinal column just above the level of the diaphragm. For gain calibration, a flow probe (1RB; Transonic, Ithaca, NY) was placed around the aorta. All signals were digitized at 2 KHz and stored on disk for off-line analysis using custom-written software. The following indices were used: ejection fraction, cardiac output, end-systolic volume, end-diastolic volume (EDV), end-diastolic and end-systolic pressures, maximal rate of pressure development (dP/dtmax), preload-recruitable stroke work (PRSW),20
preload-adjusted maximal power (powermax/EDV),21
diastolic stiffness coefficient (ß),22
time constant of isovolumic pressure decay (
D),23
and preload-adjusted peak filling rate (PFR/EDV).
Statistical Analyses
The Mann-Whitney U-test was used to compare gross severity scores and Student’s t-test was used for other comparisons. Associations between cell populations and hemodynamic indices were assessed using linear regression. The data were analyzed using SigmaStat 3.01 (SPSS Inc., Chicago, IL) software. P values of 
0.05 were considered statistically significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We first applied flow cytometry to assess cardiac inflammation in CM peptide-immunized A/J mice, which are highly susceptible to the induction of severe EAM.6,24
For the analysis, only the events with relatively high forward scatter were selected using forward/side scatter plots to exclude cell debris, red blood cells, and cells in advanced apoptosis as demonstrated by the live cell gate in Figure 1A
. CD45, a panleukocyte marker, was used to estimate percentages of heart-infiltrating leukocytes (Figure 1
; B, E, and F). The live cell gate was used to estimate percentages of leukocytes or other subsets of infiltrating cells of total live cells (Figure 1, B and C
, respectively). To estimate proportions of cell subsets out of all leukocytes, events from the live cell gate were further gated on CD45+ population as illustrated by the arrows in Figure 1
; A, B, and D.
Using CD45 as a marker, we found that immunized A/J mice had significantly higher percentages of heart-infiltrating leukocytes compared to the unimmunized controls (Figure 2C)
. Flow cytometry discriminated among mice with the same gross score (Figure 2E)
, providing a more sensitive assessment of inflammation severity. We also examined the individual subsets of the total pool of CD45+ infiltrating cells: total T cells (CD3+), helper T cells (CD4+), cytotoxic T cells (CD8+), B cells (CD19+), macrophages (CD11b+Gr1low), and neutrophils (CD11b+Gr1high). Percentages of all inflammatory cell subsets of total live cells increased with disease, with a particularly pronounced increase in CD3+ T cells (P = 0.007, Table 1
) and a relative decrease in the percentage of CD45- population, which presumably includes mainly endothelial cells along with other types of resident cells. The proportion of these populations among total infiltrating leukocytes also changed on immunization, including increases in proportions of T lymphocytes (P < 0.001) and neutrophils and a moderate decrease in the proportion of macrophages. The latter population, however, still represented the largest fraction of total leukocytes. We also found that a greater percentage of infiltrating cells expressed MHC class II in EAM compared to the unimmunized control (P < 0.001, Table 1
).
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Once the method had been established in a highly susceptible A/J mouse, we studied myocardial inflammation in BALB/c mice, which are moderately susceptible but commonly used because of the greater availability of genetically modified strains.25-27
Unimmunized BALB/c mice did not differ from unimmunized A/J mice in terms of the presence of different leukocytic subtypes in the heart (Table 1)
. In accord with our previous observations,25
BALB/c mice developed mild myocarditis undetectable by gross examination of the hearts at early time points (Figure 2B)
. During peak inflammation in the heart (approximately day 21 after immunization), these mice typically develop disease with a 50% prevalence based on light microscopic examination with a median histological score among diseased animals of 1 (on a scale from 0 to 5). More than half of the immunized mice had increased percentages of total leukocytes (>15%) on days 15 and 22 to 24, but this change did not reach statistical significance compared to the unimmunized mice (Table 1
and Figure 2D
). BALB/c mice had significantly less severe myocardial inflammation on days 30 to 34 compared to the same time point in A/J mice, as assessed by CD45+ population, and significantly lower gross scores (P = 0.002). At this stage of disease, BALB/c mice also had significantly lower proportions of CD3+ T cells and significantly higher proportions of CD11b+Gr1low macrophages out of CD45+ leukocytes (Table 1)
.
CM immunization changed the quality of the infiltrating cells in BALB/c mice with significantly greater proportions of T cells and neutrophils (P = 0.001 and P < 0.001, respectively, on day 15) (Table 1)
. The proportion of CD11b+Gr1low macrophages significantly decreased by day 15 (P = 0.03) but then increased during the acute phase and dropped again during the chronic phase. Interestingly, the chronic phase of disease (days 58 to 61) was characterized by very few total CD45+-infiltrating cells in the heart with the majority of mice having fewer CD45+ cells than the unimmunized controls (Figure 2D)
, while the severity of gross pathology increased (Figure 2B)
. Overall, BALB/c mice exhibited no association between gross scores and percentages of infiltrating leukocytes (Figure 2F)
. Importantly, on days 58 to 61 the quality of the infiltrate was different from that of the unimmunized mice and was characterized by an increased proportion of T cells. At all time points, the proportion of CD4+ T cells remained greater than that of CD8+ T cells. However, the CD4/CD8 T-cell ratio decreased with disease progression and at later time points was significantly lower compared to that in unimmunized controls (Table 1)
. At all time points of EAM, CD3+ T cells expressed higher levels of CD44, exhibiting a more activated state compared to unimmunized mice (Figure 3)
.
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, but not CD19+ B cells, of total live cells significantly correlated with the severity of inflammation, as assessed by percentages of infiltrating CD45+ leukocytes, on days 22 to 24 after immunization. However, only the proportion of neutrophils, but not any other cell type, of leukocytes had significant positive association with severity in both BALB/c and A/J mice (Figure 4
; B, C, E, and F).
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We examined the association between phenotypes of infiltrating cells and cardiac function using flow cytometry and pressure-volume relations16
on the same BALB/c mice during the chronic phase of EAM (days 58 to 61). This time point was chosen because it was associated with greater functional abnormalities compared to earlier time points (our unpublished observations). The chronic phase was characterized by increased heart weights, increases in left-ventricular volumes, and deterioration of both systolic (reduced ejection fraction, dP/dtmax, and PRSW) and diastolic (increased ß and prolonged D) performance (Table 2)
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; A to D). The range of cardiac performance during the chronic phase is shown in Figure 5, E and F
, and represents hearts with the lowest and highest proportions of CD4+ T cells. The proportion of CD4+ T cells, however, did not correlate with diastolic indices D or ß.
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D or ß (data not shown). |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Reproducibility and sensitivity of the method was demonstrated in unimmunized mice, which had similar percentages of total leukocytes with comparable proportions of different inflammatory subsets. Using flow cytometry, we confirmed our previous observations that A/J mice develop more severe acute and subacute myocarditis compared to BALB/c mice and our data corroborate earlier findings that the inflammatory infiltrate in acute EAM is composed predominantly of macrophages, has more CD4+ than CD8+ T cells, and few B220+ B cells.7,8,32 The flow cytometric method, however, provided us with the ability to precisely quantify these differences.
One of the important findings in this report is that the severity of acute myocardial inflammation is strongly associated with neutrophil accumulation in the heart. Simultaneous staining for CD11b and Gr1 gave us an opportunity to distinguish between macrophages and neutrophils. Previous studies of EAM using immunohistochemistry used CD11b (or Mac1) as a marker of macrophages and failed to make this distinction.8 Recent in vitro studies have demonstrated the ability of neutrophils to cause free radical-mediated injury of cardiomyocytes and impair their ability to shorten.33,34 These observations combined with our findings warrant further investigation on the contribution of neutrophils to the pathogenesis of autoimmune myocarditis.
We have previously shown that eosinophils accumulate in the myocardium in severe EAM.24
In this report, we did not address whether eosinophils correlate with either overall severity of disease or cardiac dysfunction. Such assessment should be performed in the future, because eosinophils have the ability to produce local tissue damage by releasing the cytotoxic contents of their cytoplasmic granules.35,36
Unlike neutrophils, eosinophils represent a population of cells that are Gr1low.37
Their flow cytometric analysis, however, is less straightforward compared to other hematopoietic cells discussed in this report. Eosinophils fall into the same granulocyte region as neutrophils on side scatter/CD45 plot in Figure 4D
, but exhibit higher side scatter signal. The distinction based on the side and forward scatter properties is enhanced by aldehyde fixation.38
In this report, the cells were not fixed to minimize the length of the procedure given the fact that all of the steps were performed on the same day.
Notably, the percentages of CD4+ and CD8+ T cells in the heart infiltrate did not add up to the percentages of CD3+ T cells. This was a consistent finding in both unimmunized and immunized mice, as well as in A/J and BALB/c mice. CD4+ and CD8+ T cells could account for approximately half of CD3+ population; these results were confirmed by using two different types of anti-CD3 mAb. Future experiments should address the nature of this CD3+CD4-CD8- population. Double-negative T cells have been shown to exist outside thymus in adult mice; they have been ascribed a regulatory role.39
CD3+CD4-CD8- population may also represent a subset of double-negative NKT cells.40
Finally these cells may be 
T cells, which have been shown to accumulate in the myocardium during fulminant myocarditis in humans and represent between 5% and 20%, and in some cases up to 50%, of the acute inflammatory infiltrate in coxsackievirus B3-induced myocarditis in mice.41-43
Flow cytometry, unlike immunohistochemistry, is useful in assessing changes in the intensity of expression of surface markers and therefore can be used to quantify their up-regulation or down-regulation. We demonstrated that heart-infiltrating T cells in immunized mice had higher surface expression of CD44 compared to those of unimmunized mice, suggesting a more aggressive nature of T cells in the inflamed hearts because CD44 is known to be up-regulated on activated and memory, but not naïve, T cells. Furthermore, it may prove to be a useful marker of potentially pathogenic T cells or of a previous inflammation. In this regard it is noteworthy that T cells on days 58 to 61 after immunization still expressed increased levels of CD44.
To our knowledge, this is the first report of a comprehensive characterization of the inflammatory infiltrate during both the acute and chronic phases of EAM in mice. We found that by days 58 to 61 after immunization very few CD45+ cells were left in the heart; however, the changes in the proportions of individual inflammatory cells remained. Despite the reduction in percentages of infiltrating leukocytes, the chronic phase was associated with increased gross pathology. These higher gross scores may reflect cardiac remodeling, such as fibrosis, which is typical of late stages of EAM, and therefore these scores may not represent myocardial inflammation per se.
During the chronic phase, percentages of total infiltrating leukocytes did not correlate with cardiac dysfunction, but proportions of CD4+ T cells in the heart infiltrate significantly correlated with systolic dysfunction and large left-ventricular volumes, major hallmarks of dilated cardiomyopathy. The importance of CD4+ T cells in disease initiation in EAM was demonstrated by T-cell depletion in A/J mice and adoptive transfer studies from C.B-17 mice with myocarditis to SCID mice.44 The role of CD8+ T cells in EAM is less well defined. It has been demonstrated that CD8 deficiency in B10.Br mice leads to exacerbation of CM-induced myocarditis.8 However, depletion of CD8+ T cells with a mAb reduced the severity of EAM in A.SW mice.32,45 The ability of CD8+ T cells to induce myocarditis was demonstrated in mice transgenically expressing an ovalbumin peptide in the heart under cardiac-specific promoter.46 These mice developed severe myocarditis on the transfer of CD8+ T cells from transgenic mice that expressed TCR specific for the ovalbumin peptide.
We found that CD4+ T cells predominated over CD8+ T cells in the myocardial infiltrate not only during the acute phase in both A/J and BALB/c mice, but also during the chronic phase of myocarditis in BALB/c mice. Unlike EAM, coxsackievirus B3-induced myocarditis in BALB/c mice is characterized by the predominance of CD8+ over CD4+ T-cell population in the myocardium during the acute phase of disease.42 The contribution of different inflammatory cells in disease progression at later time points and their role in cardiac dysfunction was not previously studied. Our findings show that the proportion of CD4+ T cells in chronic EAM represents a marker of progression to dilated cardiomyopathy, but future studies are needed to determine whether CD4+ T cells play a causal role in the deterioration of cardiac function. Similarly, the pathogenic potential of other cell types, including those that correlate with disease severity during the acute phase, should be assessed in terms of their ability to produce local damage and cardiac dysfunction. In this regard, it should be mentioned that a pathogenically important cell type may appear in the myocardium before the onset of cardiac dysfunction and therefore the existing association may be missed by examining individual time points. Finally, further analysis of CD4+ T-cell subsets, including those of Th1 and Th2 phenotypes, will provide insights into their relative contribution to cardiac injury.
In summary, we successfully applied flow cytometry to study cardiac inflammation in a mouse model of EAM and demonstrated the feasibility of using invasive hemodynamic studies and subsequent cardiac digestion and flow cytometry on the same mouse to study the effects of inflammation on cardiac function. We conclude that flow cytometry on digested hearts represents a reproducible, simple, and a relatively fast method of assessing both cellular frequencies and phenotypes and its application should facilitate our understanding of the role of inflammation in heart disease.
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Footnotes |
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Supported by the National Institutes of Health (grants HL67290, HL70729, and AI51835) and a Howard Hughes Summer Research Fellowship (to A.C.R.).
M.A. and D.G. contributed equally to this work.
Accepted for publication December 5, 2003.
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5 per group. *, P < 0.05 compared to unimmunized controls (day 0), Student’s t-test. Day 0 corresponds to unimmunized age-matched BALB/c control mice. B: Overlay histogram shows CD44 expression on CD3+ T cells from a representative unimmunized mouse (thin line, shaded) and an immunized mouse on day 23 after immunization (thick line, unshaded).
